Bacteria of the genera Rhizobium, Bradyrhizobium, Sinorhizobium and Azorhizobium, collectively known as the rhizobia, form specialized organs called nodules on the roots, and sometimes stems, of legumes and fix atmospheric nitrogen within these structures. Nodule formation is a highly specialized process that is modulated by signal molecules. In general, this phase of the interaction is a two step process. Initially, plant-to-bacteria signal molecules, usually specific flavonoids or isoflavonoids, are released by roots of the host plants. In response to the plant-to-bacteria signals the microsymbiont releases bacteria-to-plant signal molecules, which are lipo-chitooligosaccharides (LCOs), so called nod factors (also nol and noe) genes very rapidly (only a few minutes after exposure) and at very low concentrations (10−7 to 10−8 M) (Peters et al., 1986). Generally this is through an interaction with nodD, which activates the common nod genes, although the situation may be more complex, as is the case in B. japonicum, where nodD1, nodD2 and nodVW are involved (Gillette & Elkan 1996; Stacey 1995). Nod genes have been identified in the rhizobia that form nitrogen fixing relationships with numbers of the Fabiaceae family (see U.S. Pat. No. 5,549,718 and references therein). Recently, the plant-to-bacteria signal molecules have been shown to promote soybean nodulation and nitrogen fixation under cool soil temperatures (CA U.S. Pat. No. 2,179,879) and increase the final soybean grain yield on average of 10% in the field and up to 40% under certain conditions. (Long, 1989; Kondorosi, 1991; Schenes et al., 1990; Boone et al, 1999). Among the products of the nod genes induced by the plant phenolic signal molecules are various enzymes involved in the synthesis of a series of lipo chitooligosaccharides (LCOs) (Spaink, 1995; Stacey, 1995). These newly synthesized LCOs act as bacterium-to-plant signals, inducing expression of many of the early nodulin genes (Long, 1989).
LCO signal molecules are composed of three to five 1-4 β linked acetylglucosamine residues with the N-acetyl group of the terminal non-reducing sugar replaced by an acyl chain. However, various modifications of the basic structure are possible and these, at least in part, determine the host specificity of rhizobia (Spaink et al., 1991; Schultze et al., 1992).
Lipo-chitiooligosaccharides are known to affect a number of host plant physiological processes. For example, they induce: root hair deformation (Spaink et al., 1991), ontogenyof compete nodule structures (Fisher and Long, 1992; Denarie and Cullimore, 1993), cortical cell division (Sanjuan et al., 1992; Schlaman et al., 1997) and the expression of host nodulin genes essential for infection thread formation (Horvath et al., 1993; Pichon et al., 1993, Minami et al., 1996). LCOs have also been shown to activate defense-related enzymes (Inui et al., 1997). These bacterium-to-plant signals exert a powerful influence over the plant genome and, when added in the absence of the bacteria, can induce the formation of root nodules (Truchet et al., 1991). Thus, the bacteria-to-plant signals can, without the bacteria, induce all the gene activity for nodule organogenesis (Denarie et al., 1996; Heidstra & Bisseling, 1996). Moreover, the above-mentioned activities induced by LCOs can be produced by concentrations as low as 10−14 M (Stokkermans et al. 1995). The mutual exchange of signals between the bacteria and the plant are essential for the symbiotic interaction. Rhizobia mutants unable to synthesize LCOs will not form nodules. Analysis of the B. japonicum nod genes indicates that ability to induce soybean nodulation requires at least: 1) a basic tetrameric Nod factor requiring only nodABC genes or 2) a pentameric LCO (C18:1, C16:0 or C16: fatty acid and a methyl-fucose at the reducing end, sometimes acetylated) requiring nodABCZ genes (Stokkermans et al. 1995).
When added to the appropriate legume, LCOs can cause the induction of nodule meristems (Denarie et al., 1996), and therefore cell division activity. LCOs have also been shown to induce cell cycle activities in an in vitro system: (a carrot embryogenesis system) at levels as low as 10−14 M (De Jong et al. 1993).
A chemical structure of lipo chitooligosaccharides, also termed “symbiotic Nod signals” or “Nod factor”, has been described in U.S. Pat. Nos. 5,549,718 and 5,175,149. These Nod factors have the properties of a lectin ligand or lipo-oligosaccharide substances which can be purified from bacteria or synthesized or produced by genetic engineering.
The process of N2 fixation is energy intensive requiring about 10-20% of the carbon fixed by the plant. It has been estimated that an average of about 6 mg of carbon is required per mg of nitrogen fixed (Vance and Heichel, 1991). Enhanced photosynthesis, due to the Bradyrhizobium-soybean association has been previously reported. Imsande (1989a,b) reported enhanced net photosynthesis and grain yield in soybean inoculated with Bradyrhizobium japonicum compared with plants that were not inoculated but adequately supplemented with N fertilizer. Recently, Phillips et al., (1999) showed that lumichrome might act as a signal molecule in the rhizosphere of alfalfa plants, leading to increased respiration and net carbon assimilation during early stages of the Sinorhizobium meliloti-alfalfa symbiosis.
Methods to increase plant dry matter accumulation and yield are essential as world population is projected to increase by 4 billion (66%) during the next fifty years (United Nations, Population Division, 1998). In the last fifty years world crop output increased by 2.5 fold, with little increase the area of land cropped (Hoisington et al., 1999). Given the projected increase in world population we must provide another 2.5 fold increase during the next 50 years if everyone is to have reasonably reliable access to food (James, 1997). However, the primary causes of increased food production during the last 50 years (increases in harvest index, the amount of land under irrigation and the use of fertilizers, particularly N fertilizer) are largely exhausted. A century of plant breeding has resulted in little or no increase in the photosynthetic rates of most crop plants (Moss and Musgrave, 1971; Evans 1975, 1980). There thus remains a tremendous need to increase the photosynthetic rates and growth of crop plants. There also remains a need to increase production of crop plants.
There have been considerable efforts to enhance photosynthesis in crop plants with a view to increase plant productivity. Makela et al., (1999) reported enhanced photosynthesis under drought and salinity stress in tomato and turnip rape following foliar application of glycinebetanine at very low concentrations. Foliar application of methanol also increased photosynthesis in a number of plants (Noumora and Benson, 1991). Johnson and Stelizer (1991) reported increased photosynthesis in loblolly pine by application of sub-lethal doses hexazinone.
While the effects of plant-to-bacteria signal molecules (i.e. isoflavones) on nodulation, nitrogen fixation, growth and protein yield of legumes, such as soybean, and on bacteria-to-plant signal molecules (LCOs) on nodulation and nitrogen fixation in legumes have been described under certain conditions, the effect of the bacteria-to-plant signal molecules on the growth of non-legumes is unknown. In fact, the role of such bacteria-to-plant signal molecules on non-legumes has yet to be reported. In addition, the effect of LCOs on processes other than nodulation of legumes has yet to be described. Moreover, while LCOs have been associated with a growth-promoting effect in the early stages of the initiation of the symbiotic relationship between plant and bacteria, it remains to be determined whether LCOs can have an effect on plants at later stages of their life cycle.
There thus remains a need to assess the effect of LCOs on plant growth and especially on later stages thereof. Moreover, there remains a need to assess whether LCO comprising compositions can have an effect on the synthetic rate and/or growth of plants in general and especially of non-legume plants.
There also remains a need to better understand the workings of the complex homeostatic system which is involved in the regulation of photosynthesis. Moreover, there remains a need to assess the role of LCOs on photosynthesis of plants.
The present invention seeks to meet these and other needs.
The present description refers to a number of documents, the contents of which are herein incorporated by reference in their entirety.